Concurrent Chemotherapy and Immunotherapy

ABSTRACT

The concurrent administration of chemotherapy and immunotherapy has been considered a contraindication because of the concern that the induced lymphopenia would ablate therapeutic efficacy of immunotherapy. Temozolomide has been shown to be an effective chemotherapeutic for patients with malignant gliomas and to deprive patients with glioblastoma (GBM) patients of this agent in order to treat with immunotherapy is controversial. Despite conventional dogma, we demonstrate that both chemotherapy and immunotherapy can be delivered concurrently without negating the effects of immunotherapy. In fact, the temozolomide induced lymphopenia may actually be synergistic with a peptide vaccine.

This application claims the benefit of U.S. provisional application60/732,741 filed Nov. 2, 2005, the entire contents of which areexpressly incorporated herein.

This invention was made with support from the U.S. government underGrant No. R01 CA097222 from the National Institutes of Health. The U.S.government therefore retains certain rights to this invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer immunotherapy. Inparticular, it relates to enhancing response to tumor vaccines.

BACKGROUND OF THE INVENTION

Despite aggressive surgical resection, high-dose focused radiationtherapy, and chemotherapy, patients diagnosed with GBM have a mediansurvival of less than 15 months after diagnosis (Stupp et al., Optimalrole of temozolomide in the treatment of malignant gliomas. Curr NeurolNeurosci Rep. 2005 May; 5(3):198-206.). Failure of therapy can beattributed, at least in part, to a relatively narrow therapeutic indexso that attempts at dose escalation results in dose-limiting systemic orneurological toxicity. The use of immunotherapy has held promise for thepotential treatment of these tumors but until recently, few havedemonstrated clinical efficacy. Several clinical trials, with selectedpatients, involving vaccination of glioma patients with dendritic cells(DCs) and either acid-eluted peptides (Ashkenazi et al., A selectiveimpairment of the IL-2 system in lymphocytes of patients withglioblastomas: increased level of soluble IL-2R and reduced proteintyrosine phosphorylation. Neuroimmunomodulation. 1997; Kolenko et al.,Tumor-induced suppression of T lymphocyte proliferation coincides withinhibition of Jak3 expression and IL-2 receptor signaling: role ofsoluble products from human renal cell carcinomas. J Immunol. 1997 Sep.15; 159(6):3057-67; Liau et al., Dendritic cell vaccination inglioblastoma patients induces systemic and intracranial T-cell responsesmodulated by the local central nervous system tumor microenvironment.Clin Cancer Res. 2005 Aug. 1; 11(15):5515-25) or an antigen-specificpeptide (Heimberger A B, Archer G E, et al., Dendritic cells pulsed witha tumor-specific peptide induce long-lasting immunity and are effectiveagainst murine intracerebral melanoma. Neurosurgery. 2002 January;50(1):158-64; discussion 164-6) have demonstrated promise by increasingmedian survival time to a range of 20-31 months. Furthermore, in arecently completed phase II clinical trial utilizing an antigen-specificimmunotherapeutic approach, time to progression (TTP) in GBM patientswas delayed to 15 months, which is in marked contrast to the standard ofcare consisting of radiotherapy and temozolomide that had a TTP of 7months (Stupp et al., 2005, supra), and median survival was 29 months(Heimberger et al, J Transl Med. 2005 Oct. 19; 3:38 The natural historyof EGFR and EGFRvIII in glioblastoma patients.). Cumulatively, theseimmunotherapy trials suggest that despite the inherent immunosuppressionof malignant glioma patients, efficacious immune responses can begenerated. However, there is reluctance to not treat GBM patients withsome form of chemotherapy given the recently established standard ofcare and the overall poor prognosis.

There is a continuing need in the art to develop better methods fortreating tumors in general and glioblastomas in particular.

SUMMARY OF THE INVENTION

A method is provided for treating a tumor in a subject. Atreatment-effective amount of an EGFRvIII peptide and atreatment-effective amount of a chemotherapeutic agent which induceslymphopenia are administered to the subject.

According to another embodiment a method is provided for treating atumor in a subject. A treatment-effective amount of an EGFRvIII peptideconjugated to KLH is administered to the subject with the tumor.Granulocyte/macrophage colony stimulating factor (GM-CSF) is alsoadministered as an adjuvant in an effective amount concurrently with theEGFRvIII peptide. A treatment-effective amount of an alkylating agent isalso administered to the subject.

According to still another embodiment, a method is provided for treatinga tumor in a subject. A treatment-effective amount of an anti-tumorvaccine and a treatment-effective amount of temozolomide or apharmaceutically acceptable salt thereof are administered to thesubject.

According to still another embodiment, a method is provided for treatinga tumor in a subject. A treatment-effective amount of an anti-tumorvaccine and a treatment-effective amount of a chemotherapeutic agentwhich induces lymphopenia are administered to the subject.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with additionalmethods for treating treatment-refractory tumors.

DETAILED DESCRIPTION OF THE INVENTION

The concurrent administration of chemotherapy and immunotherapy has beenconsidered a contraindication because of the concern that thechemotherapy-induced lymphopenia would ablate therapeutic efficacy ofimmunotherapy. Temozolomide has been shown to be an effectivechemotherapeutic for patients with malignant gliomas and to deprivepatients with glioblastoma (GBM) patients of this agent in order totreat with immunotherapy is controversial. Despite conventional dogma,the inventors demonstrate that both chemotherapy and immunotherapy canbe delivered concurrently without negating the effects of immunotherapy.In fact, the temozolomide induced lymphopenia may actually besynergistic with a peptide vaccine. Although applicants do not wish tobe bound by an particular theory regarding mechanism of action, theobserved synergy may be secondary to inhibition of Tregs or the failureto recover of Tregs, which permits an increase of effector cytotoxicCD8⁺ T cells. Other mechanisms may also be involved.

“EGFRvIII” or “Epidermal Growth Factor Receptor mutation III” is a knownmutant form of the Epidermal Growth Factor Receptor. See, e.g., U.S.Pat. No. 6,503,503; see also U.S. Pat. Nos. 6,900,221; 6,673,602;6,479,286; and 6,129,915. The mutation which causes the production ofthe vIII protein is typically characterized by a consistent andtumor-specific in-frame deletion of 801 base pairs from theextracellular domain that splits a codon and produces a novel glycine atthe fusion junction.

“EGFRvIII peptide” as used herein refers to a peptide of suitablelength, e.g., at least 10 or 12 amino acids, and up to 16, 20 or 30amino acids, or more, which spans the mutated splice junction of thecorresponding EGFRvIII protein. Examples include but are not limited to:H-LEEKKGNYVVTDHS-OH, or “PEP-3.” The EGFRvIII peptide may be from (orcorrespond in sequence to) the EGFRvIII of any mammalian species, but ispreferably human. Particular wild-type sequences of EGFR are shown inSEQ ID NO: 6 to 9.

“Carrier protein” as used herein refers to a protein which does notpossess high homology to a protein found in the species that isreceiving a composition of the invention and elicits an immune response.A protein possesses high homology if it is at least 75% identical, morepreferably at least 85% identical or at least 90% identical to a proteinas determined by any known mathematical algorithm utilized for thecomparison of two amino acid sequences (see, e.g., Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268; Karlin and Altschul,1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877; Torellis and Robotti,1994, Comput. Appl. Biosci. 10: 3-5; and Pearson and Lipman, 1988, Proc.Natl. Acad. Sci. 85: 2444-8). Preferably, the percent identity of twoamino acid sequences is determined by BLAST protein searches with theXBLAST program, score=50, word length=3. Examples of heterologouscarrier proteins include, but are not limited to, KLH, PhoE, mLT, TraT,or gD from BhV-1 virus. See, e.g., U.S. Pat. No. 6,887,472. Such carrierproteins may be conjugated or linked to the tumor antigen directly or byan intervening linker segment such as a chain of one or more (e.g., 2,4, 6) intervening amino acids (e.g., an intervening CYS residue) inaccordance with known techniques.

“KLH” or “keyhole-limpet hemocyanin” is a known carrier protein to whichanother protein may be conjugated in accordance with known techniques.See, e.g., U.S. Pat. No. 6,911,204.

“Adjuvant” as used herein refers to anyone of a diverse class ofcompounds that enhance the therapeutic efficacy of a vaccine which isadministered concurrently with the adjuvant. In some embodiments theadjuvant is a hematopoietic growth factor such as GM-CSF. Commonexamples of adjuvants include but are not limited to aluminiumhydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsionbased on, for example a mineral oil, such as Bayol Fo or Marcol 52™ or avegetable oil such as vitamin E acetate, saponins, BCG, M. vaccae,Tetanus toxoid, Diphtheria toxoid, Bordetella pertussis, interleukin 2,interleukin 12, interleukin 4, interleukin 7, Complete Freund'sAdjuvant, Incomplete Freund's Adjuvant, and a nonspecific adjuvant. See,e.g., U.S. Pat. No. 6,699,483.

“Hematopoietic growth factors” or “HGFs” are known. See, e.g., U.S. Pat.No. 6,863,885. In general, HGFs are glycoprotein cytokines that regulatethe proliferation and differentiation of hematopoietic progenitor cells.The hematopoietic growth factors intended to be used in the presentinvention can be selected from the group G-CSF (granulocyte colonystimulating factor), SCF (stem cell factor), GM-CSF (granulocytemacrophage colony stimulating factor), IL-1 (interleukin-1), IL-3, IL-6,IL-8, IL-11, IL-12, LIF (leukemia inhibitory factor), FGF-beta(fibroblast growth factor beta), FLT3, or a combination thereof. Thesegrowth factors can be purchased (e.g., R&D Systems, Minneapolis, Minn.)or made following procedures set forth in the art generally and inpublications describing the factors. Additionally, the hematopoieticgrowth factor can be a modified form of the factor or a fusion proteinof hematopoietic growth factors selected from the group GCSF, SCF,GM-CSF, IL-I, IL-3, IL-6, IL-8, IL-11, IL-12, LIF, FGF-beta, and FLT3.HGFs include modified growth factors (e.g., muteins) and fusionproteins, which can be made according to methods known in the art. See,e.g. (Sambrook et aI., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).Hematopoietic growth factors that stimulate macrophage function such asGM-CSF are particularly preferred. These can be used as adjuvants.

“External beam radiotherapy” can be carried out by delivering a beam ofhigh-energy x-rays to the location of the patient's tumor. The beam isgenerated outside the patient and is targeted at the tumor site. Noradioactive sources are placed inside the patient's body. This can beused in conjunction with any other treatment step according to theinvention.

“Treat” as used herein refers to any type of treatment or preventionthat imparts a benefit to a subject afflicted with a disease or at riskof developing the disease, including improvement in the condition of thesubject (e.g., in one or more symptoms), delay in the progression of thedisease, delay the onset of symptoms or slow the progression ofsymptoms, etc. As such, the term “treatment” also includes prophylactictreatment of the subject to prevent the onset of symptoms.

As used herein, “treatment” and “prevention” are not meant to imply cureor complete ablatement of symptoms. Rather, these refer to any type oftreatment that imparts a benefit to a patient afflicted with a disease,including improvement in the condition of the patient (e.g., in one ormore symptoms), delay in the progression of the disease, etc.

“Treatment-effective amount” as used herein means an amount of theantibody sufficient to produce a desirable effect upon a patientinflicted with cancer such as gliomblastoma, including improvement inthe condition of the patient (e.g., in one or more symptoms), delay inthe progression of the disease, etc.

Subjects in need of treatment by the methods described herein includesubjects afflicted with glioblastoma or astrocytoma, as well as subjectsafflicted with other solid tumors or cancers such as lung, colon,breast, brain, liver, prostate, spleen, muscle, ovary, pancreas, headand neck, skin (including melanoma), etc. Subjects in need of treatmentparticularly include subjects afflicted with a tumor, such as a braintumor, that expresses EGFRvIII. The tumor may be a primary tumor, ametastatic tumor, or a recurrent tumor. Subjects to be treated by themethods of the invention particularly include subjects afflicted with atumor expressing EGFRvIII, including gliomas, fibrosarcomas,osteosarcomas, melanoma, Wilms tumor, colon carcinoma, mammary and lungcarcinomas, and squamous carcinomas. Subjects to be treated by thepresent invention most particularly include subjects afflicted withbrain tumors or cancers, such as glioblastomas, particularlyglioblastoma multiforme, and cystic astrocytoma.

The present invention is primarily concerned with the treatment of humansubjects, including male and female subjects and neonatal, infant,juvenile, adolescent, adult, and geriatric subjects, but the inventionmay also be carried out on animal subjects, particularly mammaliansubjects such as mice, rats, dogs, cats, livestock and horses forveterinary purposes, and for drug screening and drug developmentpurposes.

The pharmaceutical compositions of the invention can be prepared inaccordance with known techniques. Typically, the active agents areincluded in a pharmaceutically acceptable carrier. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.9% saline, 0.3%glycine, hyaluronic acid and the like. These compositions may besterilized by conventional, well known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

The compositions and methods of the invention may include theadministration of one or more co-adjuvants. Suitable co-adjuvantsinclude, but are not limited to: (1) aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2)oil-inwater emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) MF59 (PCTPublication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and0.5% Span 85 formulated into submicron particles, (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™) (for a further discussion of suitable submicron oil-in-wateremulsions for use herein, see PCT Publication No. WO 99/30739, publishedJun. 24, 1999); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particle generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreunds Adjuvant (CF A) and Incomplete Freunds Adjuvant (IF A); (5)cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63 (where lysine is substituted for thewild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-SI09 (whereserine is substituted for the wild-type amino acid at position 109),adjuvants derived from the CpG family of molecules, CpG dinucleotidesand synthetic oligonucleotides which comprise CpG motifs (see, e.g.,Krieg et aI., Nature, 374:546 (1995) and Davis et aI., J. Immunol.,160:870-876 (1998)) and PT-K9/GI29 (where lysine is substituted for thewild-type amino acid at position 9 and glycine substituted at position129) (see, e.g., PCT Publication Nos. WO93/13202 and WO92/19265); (7)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition. See, e.g., U.S. Pat. No. 6,534,064;and (8) other ligands for Toll-like receptors in addition to CpG and RIMadjuvants, such as bacterial flagellin (an effective adjuvant for CD4+ Tcells; see IJ Immunol. 169: 3914-9 (October 2002).

The active agents may be administered by any medically appropriateprocedure, e.g., normal intravenous or intra-arterial administration,injection into the cerebrospinal fluid). In certain cases, intradermal,intracavity, intrathecal or direct administration to the tumor or to anartery supplying the tumor is advantageous. Where the tumor or a portionthereof has been previously surgically removed the treatment agents maybe administered into the site of the tumor (and particularly into anenclosed cavity or “resection cavity” at the site of the tumor) bydirect injection or through a pre-implanted reservoir.

Dosage of the active agents will depend on, among other things, thecondition of the subject, the particular category or type of cancerbeing treated, the route of administration, the nature of thetherapeutic agent employed, and the sensitivity of the tumor to theparticular therapeutic agent.

In general, the dose of the tumor antigen or vaccine, such as EGFRvIII,including any carrier protein or peptide conjugated thereto, will befrom 10, 100 or 500 μg up to 2 or 3 mg per subject, for each dose. Dosesmay be given on a single occasion, optionally including follow-up or“booster” doses (e.g., one, two or three follow up or “booster” dosagesgiven at intervals of from one to three weeks). Note that doses can bedivided, such as administering to different injection sites, to reduceside effects such as local responses, if desired. Where the formulationcontains both tumor antigen bound (or “conjugated”) to the carrierprotein and tumor antigen free of the carrier protein, the calculateddosage can include both the amount of both bound and free tumor antigenand carrier protein.

In general, the dose of the adjuvant such as GM-CSF will also be from 10or 20 μg up to 500 μg, or 1 or 2 mg per subject, administered on thesame schedule or different schedule from the dose of the tumor antigen.When administered on the same schedule the adjuvant may be administeredin the same carrier as the tumor antigen. When not combined in the samecarrier, the dose of adjuvant need only be administered sufficientlyclose in time to the dose of tumor antigen to enhance the efficacythereof (e.g., within one or two hours; on the same day; etc.).

Alkylating agents useful for carrying out the present invention include(but are not limited to) 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) andtetrazine derivatives, particularly[3H]imidazo[5,1-d]-1,2,3,5-tetrazin-4one derivatives such astemozolomide and analogs thereof (including pharmaceutically acceptablesalts and pro drugs thereof). Such compounds are known. See, e.g., U.S.Pat. Nos. 6,096,724; 6,844,434; and 5,260,291. Examples of alkylatingagents useful for carrying out the present invention include[3H]imidazo[5,1-d]-1,2,3,5-tetrazin-4-ones alkylating agents,particularly those of the general formula:

wherein R¹ represents a hydrogen atom, or a straight- or branched-chainalkyl, alkenyl or alkynyl group containing up to 6 carbon atoms, eachsuch group being unsubstituted or substituted by from one to threesubstituents selected from halogen (i.e. bromine, iodine or, preferably,chlorine or fluorine) atoms, straight- or branched-chain alkoxy, (e.g.methoxy), alkylthio, alkylsullihinyl and alkylsulphonyl groupscontaining up to 4 carbon atoms, and optionally substituted phenylgroups, or R¹ represents a cycloalkyl group, and R² represents acarbamoyl group which may canyon the nitrogen atom one or two groupsselected from straight- and branched-chain alkyl and alkenyl groups,each containing up to 4 carbon atoms, and cycloalkyl groups, e.g., amethylcarbamoyl or dimethylcarbamoyl group. When the symbol R¹represents an alkyl, alkenyl or alkynyl group substituted by two orthree halogen atoms, the aforesaid halogen atoms may be the same ordifferent. When the symbol R¹ represents an alkyl, alkenyl or alkynylgroup substituted by one, two or three optionally substituted phenylgroups the optional substituents on the phenyl radical(s) may beselected from, for example, alkoxy and alkyl groups containing up to 4carbon atoms (e.g. methoxy and/or methyl group(s)) and the nitro group;the symbol R¹ may represent, for example, a benzyl or p-methoxybenzylgroup. Cycloalkyl groups within the definitions of symbols R¹ and R²contain 3 to 8, preferably 6, carbon atoms. The compounds may beprovided as salts or prodrugs, particularly alkali metal salts when R¹is H. See, e.g., U.S. Pat. No. 5,260,291.

Temozolomide, in oral dosage form as 5 mg, 20 mg, 100 mg, and 250 mgcapsules, is commercially available as TEMODAR™ from ScheringCorporation, Kenilworth N.J. 07033 USA.

Alkylating agents may be prepared in pharmaceutically acceptableformulations in like manner as described above, in the same or differentformulation that contains the tumor vaccine, e.g., EGFRvIII peptide.

In a preferred embodiment, the alkylating agent is administered in acycle of daily doses for 3, 4, 5, 6 or 7 consecutive days. A suitabledaily dose may be from 50, 100 or 150 mg/m²/dose, up to 200, 250 or 300mg/m²/dose. This cycle may be repeated, e.g., every two, three, four orfive weeks, for up to a total of 6, 8 or 10 cycles. The first dose inthe first cycle of alkylating agent may be administered at any suitablepoint in time. In some embodiments the first dose of alkylating agent isadministered up to two or four weeks before administration of thetherapeutic antibody; in some embodiments the first dose of alkylatingagent is administered at least two, four or six weeks following theadministration of the therapeutic antibody. Additional schedules ofadministration may be included where additional therapeutic treatmentssuch as external beam radiotherapy are also applied to the subject.

Optionally, the subject may also receive external beam radiotherapy. Forexample, external beam radiotherapy may be utilized for brain tumorssuch as glioblastoma. External beam radiotherapy is known and can becarried out in accordance with known techniques. The beam can begenerated by any suitable means, including medical linear acceleratorsand Cobalt 60 external beam units. The radiation source can be mountedin a gantry that rotates around the patient so that a target area withinthe patient is irradiated from different directions. Before irradiationthe treatment is typically planned on a computer using algorithms thatsimulate the radiation beams and allow the medical personnel to designthe beam therapy. Numerous variations of external beam therapy that canbe used to carry out the present invention will be readily apparent tothose skilled in the art. See, e.g., U.S. Pat. Nos. 6,882,702;6,879,659; 6,865,253; 6,863,704; 6,826,254; 6,792,074; 6,714,620; and5,528,650.

External beam therapy is preferably administered in a series of sessionsin accordance with known techniques, with the sessions preferablybeginning two to four weeks after administration of the therapeuticantibody. For example, the external beam radiotherapy may beadministered 3, 4, 5, 6 or 7 days a week, over a period of four, five,six or seven weeks, at a daily dose of 0.5 or 1 Gy, up to 2 or 3 Gy,until the total desired dose (e.g., 30 or 40 Gy, up to 50 or 60 Gy) isadministered.

The delivered dose may be to an area including a margin of normal tissue(e.g., ˜1, 2 or 3 cm margin in all directions) around the tumor, orwhere the tumor or a portion thereof has previously been surgicallyremoved, around the site of the tumor.

Where external beam radiotherapy is employed, the patient may receive anadditional schedule of chemotherapeutic agent administration, differentfrom that described above, at a somewhat lower dose, during the courseof the radiotherapy. For example, the patient may receive daily doses ofchemotherapeutic agent, e.g., alkylating agent in an amount of from 25or 50 mg/m²/dose up to 100 or 125 mg/m²/dose daily during the course ofthe external beam therapy.

Examples of tumor antigens which can be used as anti-tumor vaccinesinclude but are not limited to Cyclin-dependent kinase 4; β-catenin;Caspase-8; MAGE-1; MAGE-3; Tyrosinase; Surface Ig idiotype; Her-2/neuReceptor; MUC-1; HPV E6 and E7; CD5 Idiotype CAMPATH-1, CD20; Cellsurface glycoprotein CEA, mucin-1; Cell surface carbohydrate Lewis^(x);CA-125; Epidermal growth factor receptor; p185HER2; IL-2R; FAP-α;Tenascin; and metalloproteinases. EGFRvIII is exemplary oftumor-specific antigens. Cells which express these antigens can also beused as vaccines. Preferably the cells are killed prior toadministration. The cells can be fractionated so that a fractionenriched for the tumor antigen is used as a vaccine. These antigens aremerely exemplary and are not intended to be a comprehensive of the manyuseful antigens known in the art or which may be used.

Multiple preclinical model systems have demonstrated that the depletionof immune cell subsets can abrogate the efficacy of several types ofimmunotherapeutic approaches (Heimberger et al., 2003) indicating thatchemotherapy administered during the effector stages of immunotherapymay be deleterious to efficacy. However, this does not precludeutilizing these agents together when appropriately timed to minimize theaforementioned effects. Furthermore, although applicants do not wish tobe bound by any particular theory regarding mechanism of action, thedepletion of certain effector cells, such as Tregs, may be a highlydesirable outcome of chemotherapy yielding greater immunotherapeuticefficacy or may promote a desirable cytokine profile for adequate tumorcontrol.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1

To test the hypothesis that chemotherapy and immunotherapy can beadministered concurrently, we treated a patient with a newly diagnosedGBM using the standard of care, temozolomide, while also administering apeptide vaccine targeting the epidermal growth factor variant III(EGFRvIII) (Heimberger et al., 2006). The amplification of the epidermalgrowth factor receptor (EGFR) gene, which results in over expression ofthe EGFR, a transmembrane tyrosine kinase receptor (Ekstrand et al.,1991) is associated with the mutant EGFR gene, EGFRvIII (Wikstrand etal., 1997). Previous work has shown that EGFR amplification is evidentin all GBMs expressing EGFRvIII (Heimberger et al., 2005) and GBMslacking the amplified EGFR are not positive for EGFRvIII protein (Aldapeet al., 2004).

In May of 2005, a 51-year-old Caucasian man was evaluated followingcomplaints of a three-week history of persistent morning headacheswithout associated nausea. A magnetic resonance (MR) image revealed amulti-lobular, irregularly enhancing lesion measuring 6.6×5.3×4.3 in theanterior aspect of the right temporal lobe. The sylvian fissure wasbowed upward and there was 6 mm of midline shift. The patient underwenta gross total resection, with histology demonstrating a biphasicglioblastoma and malignant sarcoma. These components were confirmed bypositive immunohistochemistry in the glioblastoma component withglial-fibrillary astrocytic protein (GFAP) and abundant reticulinproduction in the sarcoma component. A trichrome stain confirmed thebiphasic nature of the tumor. EGFR-528 and EGFRvIII antibodyimmunohistochemistry staining was positive (Heimberger et al., 2005),with the EGFRvIII staining demonstrating strong diffuse reactivity,while the EGFR-528 staining was more focal. PTEN was strongly positiveand p53 reactivity was present in more than 30% of tumor nuclei. Themethylguanine-DNA methyltransferase (MGMT) DNA-repair gene wasmethylated (Hegi et al., 2005).

Post-operatively the patient underwent conventional external beamradiotherapy of 6000 cGy in 30 fractions. Concurrent temozolomide at 75mg/m2 was administered during radiotherapy (Stupp et al., 2005). An MRimage taken at the completion of radiotherapy was unchanged anddemonstrated no evidence of progression. The patient then underwent aleukapheresis to obtain sufficient cells for immunological monitoringpurposes. The patient received three intradermal (i.d.) injections ofPEPvIII-3 (LEEKKGNYVVTDHC), conjugated to keyhole limpet hemocyanin(KLH) at a 1:1 ratio (w/w) (PEPvIII-KLH) (500 μg/immunization) withgranulocyte-macrophage colony-stimulating factor (GM-CSF) (142μg/immunization) every two weeks over an interval of 6 weeks (theinduction phase). Thereafter, he underwent a second leukapheresis forimmunological monitoring purposes. At this point, the patient beganmaintenance cycles of temozolomide of 150 mg/m2 on day 1-5. Beginning onday 19 of each cycle, complete blood counts were monitored every otherday until there was evidence of recovery of the white blood cell countnadir. At nadir recovery, the patient received the vaccine i.d., usuallyon day 23 (range=19-25) of his 28-day cycle.

Example 2

Delayed type hypersensitivity (DTH) testing to common recall antigensand the components of the vaccine were evaluated prior to the start ofthe vaccines, after the 3rd vaccine and monthly during his maintenancecycle on day 26. Prior to the start of the vaccine and after thecompletion of radiation and concurrent temozolomide the patient was onlyreactive to Candida and had no DTH reaction to the components of thevaccine, PEPvIII or KLH. However, after the 3rd vaccination, the patientbecame responsive to the KLH component of the vaccine. After the 10thvaccination, and while receiving concurrent temozolomide, he becamereactive to the PEPvIII component of the vaccine. For comparison, of thepatients that received the vaccine without cycled temozolomide (n=22),less than 15% ever became reactive to the PEPvIII component. After themost recent follow-up and administration of the 14th vaccination, thepatient was markedly indurated (16×15 mm) at the PEPvIII DTH site. Thiswould indicate that the temozolomide did not negatively influence thedevelopment of DTH responses in this particular patient.

Example 3

To determine if PEPvIII-specific humoral responses were induced, serumwas obtained from the patient monthly and was stored at −20° C. beforeanalysis in a PEPvIII-Dynabead® assay. PEPvIII or the extracellulardomain of EGFRvIII (EGFRvIII-ECD) were covalently linked to magneticmicrospheres that were used to capture specific antibodies frompatient's serum (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. All serum samples are initially diluted1:10 with phosphate-buffered saline (PBS)+0.5% bovine serum albumin(BSA) and assayed in triplicate. To determine specificity, an additionalsample set was pre-incubated for 15 minutes with 500 ng of the PEPvIIIpeptide to block any anti-PEPvIII that would be captured by the PEPvIIIconjugated Dynabeads. Standards of human-mouse chimeric anti-PEPvIIIantibody (81-0.11 ng/ml) are run with each assay along with positive(patient sample ACT4) and negative (normal donor serum) controls. Theflow cytometer was standardized with PE-FACS microbeads and un-reactedPEPvIII Dynabeads. Prior to the administration of the vaccine, therewere no detectable humoral responses to the EGFRvIII. After thevaccination, there was a significant increase in IgG responses toEGFRvIII to a mean fluorescent intensity (MFI) of 13 and the humoralresponses have been maintained despite administration the temozolomide.

Example 4

To determine if CD8+ cytotoxic responses were induced to PEPvIII, thepatient's peripheral blood mononuclear cells (PBMCs) from eachleukapheresis and monthly PBMCs were stimulated with either tetanustoxoid (QYIKANSKFIGITE) (SEQ ID NO: 5) (10 m/ml) (positive control),PEP-1 (HDTVYCVKGNKELE) (SEQ ID NO: 4) (10 m/mL) (negative control),PEPvIII (10 m/mL) (vaccine component), or KLH (10 μg/ml) (vaccinecomponent). A negative control included un-stimulated cells. Thecorresponding isotype controls were used for each condition, includingγ-interferon (IFN) secretion. All wells were incubated for 6 hr at 37°C. with Golgiplug™ (Pharmingen, San Diego, Calif.), a protein transportinhibitor that blocks the intracellular transport process. Afterincubation, the cells were washed and blocked for non-specific bindingusing purified anti-CD16 antibody (Pharmingen) and rabbit serum(Pharmingen). The cells were stained for surface markers (CD3, CD4, CD8)by incubating with the appropriate fluorescein-isothiocyanate andallophycocyanin labeled fluorescence-labeled primary antibody or isotypecontrol (Pharmingen). Cells were then fixed with Cytofix/Cytoperm (BDBiosciences, San Jose, Calif.) and then incubated withphycoerythrin-labeled antibody against γ-IFN or the isotype control.After staining, cells were washed and a minimum of 1×10⁵ live, gatedevents were assessed by flow cytometry on a FACSCalibur flow cytometerusing Cellquest software (BD Immunocytometry systems, San Jose, Calif.).Prior to receiving the vaccine the patient had minimal response in theun-stimulated controls and with the PEP-1 negative control. Afterreceiving the vaccine, and during administration of the temozolomide,there was an increase in PEPvIII-specific γ-IFN producing CD8⁺ T cells.

Example 5

To characterize the response of the various T cell populations during acycle of temozolomide (5/21 schedule) and concurrently administeredvaccine (day 19 on this example), we obtained peripheral blood on days0, 3, 5, 12, 19, 23, 25 and 26. By flow analysis cytometry, weinvestigated the percentage of the CD8+ T cell and CD4+CD25+FoxP3+regulatory T cells subsets during an immunochemotherapy cycle. Allfluorescence-conjugated monoclonal antibodies (mAb) (PerCP-Cy5.5-CD3,FITC-CD8, APC-CD4 and PE-CD25) were purchased from BD Biosciences exceptthe FITC-labeled mAb of FoxP3 was made by eBioscience. The surface andintracellular staining of peripheral blood cells were performedaccording to the standard procedures provided by the manufacturer.Results were analyzed by FACSCalibur flow cytometer using Cellquest Prosoftware (BD Biosciences). In contrast to the decline of the CD8+ T cellsubset, the Treg population started to increase after the administrationof temozolomide for 3 days and reached its peak (0.9% of total CD4⁺ Tcells) on day 12. The Tregs then began to drop until day 23 while theCD8⁺ T cell numbers started to recover. At the end of the course, bothof CD8⁺ T cell and Treg populations recovered to pre-treatment levels.The vaccination resulted in a boost of CD8+ cytotoxic T cells during aperiod of relative diminished Tregs.

Example 6

Over the last 15 months, the patient underwent complete physicalexamination and brain MR imaging at two-month intervals. His exam hasremained stable and MR imaging has failed to demonstrate any evidence ofrecurrence. He works full time without impairment and has a Karnofskyperformance status (KPS) of 100% and mini-mental status exam score of30/30. His neurological exam is completely normal.

This report suggests that concurrent administration of chemotherapy withimmunotherapy may be possible if the timing of the treatments arecarefully monitored. In the case reported, there are several findingsthat indicate that the co-administration of the temozolomide has notaffected the efficacy of the PEPvIII-KLH vaccine. First, the patient hasnot yet progressed at 15 months of follow-up. This was the median TTPfor patients (n=22) that received only vaccination therapy. Thus, theclinical efficacy does not appear to have been effected compared topatients that did not receive the concurrently administeredtemozolomide. The patient developed DTH responses to the PEPvIIIcomponent of the vaccine, even while receiving temozolomide, whereasonly 15% of the patients receiving the vaccine alone developed thesetypes of responses. Furthermore, the area of PEPvIII DTH reactivity hascontinued to increase with subsequent vaccinations. Third, IgG specificresponses to PEPvIII were induced after the 3rd vaccination and havebeen maintained while receiving the concurrent temozolomide. Fourth, theinduced PEP-3 specific CD3⁺CD8⁺γ-IFN producing T cells do not appear tobe diminished during cycles of concurrently administered temozolomidebut appear enhanced during the concurrently administered temozolomide.Finally, we have followed the CD8⁺ T cell and Treg populations during asingle treatment cycle and found that there appears to be a window of Teffector (CD8⁺ T cell) responsiveness with a relative diminution ofTregs. Thus, the concurrent administration of temozolomide and vaccinedoes not appear to diminish the induced immune responses, in the mannerin which we have described.

The use of lymphodepletion to augment immunological responses has beendescribed in both murine model systems (Berenson et al., 1975; Cheeveret al., 1980; North, 1982) and in human cancer patients (Dudley et al.,2002; Dudley et al., 2005). Multiple mechanisms have been proposed to beresponsible for these enhanced anti-tumor responses. Lymphodepletion mayremove competition at the surface of antigen presenting cells (Kedl etal., 2000), enhance the availability of cytokines such as IL-7 andIL-15, which augment T cell activity (Gattinoni et al., 2005) anddeplete the immune inhibitory Tregs (Anthony et al., 2005). Chemotherapycould also potentially augment immunological responsiveness by enhancingimmune priming and presentation (Nowak et al., 2002), enhancing antigenexpression (Aquino et al., 1998), and enhancing targets for immuneeradication (Ciusani et al., 2002). When a vaccination is administeredduring the nadir of temozolomide, we hypothesized that there may be anenhanced effector response. Those effector responses may be secondary toa lag in the recovery of Tregs thus allowing a greater clonotypicexpansion than would have otherwise been seen without the temozolomide.This was certainly observed during a monitored chemoimmunotherapy cycleon this particular patient. The lag of recovery of Tregs relative toeffector T cells is not surprising given the normal physiological rolesof immune cell responses. In order to mount an immune response, Teffectors would need to become activated, proliferate and mediate theirresponse. If this remained unchecked by homeostatic mechanisms such asTregs, then the T cell proliferation would escalate unabated. Therefore,the delay of Treg response would allow for efficacious immune responsesbut eventual down-modulation/regulation of this response.

In conclusion, this case report suggests that co-administration ofchemotherapy and immunotherapy may not be deleterious.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   Hatano, M., J. Eguchi, et al. (2005). “EphA2 as a glioma-associated    antigen: a novel target for glioma vaccines.” Neoplasia 7(8):    717-22.-   Liu, G., J. S. Yu, et al. (2004). “AIM-2: a novel tumor antigen is    expressed and presented by human glioma cells.” J Immunother 27(3):    220-6.-   Liu, M., B. Dai, et al. (2006). “FoxM1B is overexpressed in human    gliobastomas and critically regulates the tumorigenicity of glioma    cells.” Can Res 66(7): 3593-3602.-   Xie, D., Y. X. Zeng, et al. (2006). “Expression of cytoplasmic and    nuclear survivin in primary and secondary human glioblastoma.” Br J    Cancer 94(1): 108-114.

We claim:
 1. A method of treating a tumor expressing EGFRvIII in asubject, comprising the steps of: administering to the subject an amountof an EGFRvIII peptide effective to induce an IgG response to EGFRvIIIpeptide or to induce EGFRvIII peptide-specific γ-IFN producing CD8⁺ Tcells, after administering an amount of temozolomide or apharmaceutically acceptable salt thereof effective to induce lymphopeniaof CD8+ T cells; wherein the combined administration of the peptide andthe temozolomide or pharmaceutically acceptable salt thereof increasesimmunotherapeutic efficacy.
 2. The method of claim 1 wherein theEGFRvIII peptide is conjugated to keyhole limpet hemocyanin (KLH). 3.The method of claim 1 further comprising the step of: administering tothe subject GM-CSF as an adjuvant in an effective amount concurrentlywith the EGFRvIII peptide.
 4. The method of claim 1 wherein the EGFRvIIIpeptide has the sequenceLEU-GLU-GLU-LYS-LYS-GLY-ASN-TYR-VAL-VAL-THR-ASP-HIS-CYS (SEQ ID NO: 3)and wherein KLH is conjugated to the CYS residue.
 5. The method of claim4 wherein the EGFRvIII peptide is conjugated to the KLH with aheterobifunctional cross-linker.
 6. The method of claim 5 wherein theheterobifunctional cross-linker is sulfosuccinimidyl6-[3′(2-pyridyldithio)-propionamido]hexanoate.
 7. The method of claim 1wherein a treatment effective amount of temozolomide is administered. 8.The method of claim 1 wherein the tumor is a malignant glioma.
 9. Themethod of claim 1 wherein a treatment effective amount of apharmaceutically acceptable salt of temozolomide is administered.
 10. Amethod of treating a tumor expressing EGFRvIII in a subject, comprisingthe steps of: administering to the subject an amount of an EGFRvIIIpeptide conjugated to KLH effective to induce an IgG response toEGFRvIII peptide or EGFRvIII peptide-specific γ-IFN producing CD8⁺ Tcells after administering to the subject an amount of temozolomide or apharmaceutically acceptable salt thereof effective to induce lymphopeniaof CD8⁺ T cells; and administering to the subject GM-CSF as an adjuvantin an effective amount concurrently with the EGFRvIII peptide; whereinthe combined administration of the peptide and the temozolomide or thepharmaceutically acceptable salt increases immunotherapeutic efficacy.11. The method of claim 10 wherein the tumor is a malignant glioma. 12.The method of claim 10 wherein a treatment effective amount oftemozolomide is administered.
 13. The method of claim 1 wherein theEGFRvIII peptide is administered after the CD8+ T cells begin to recoverfrom nadir of the induced lymphopenia.
 14. The method of claim 10wherein the EGFRvIII peptide is administered after the CD8+ T cellsbegin to recover from nadir of the induced lymphopenia.
 15. The methodof claim 1 wherein the EGFRvIII peptide is administered after T_(REG)cells in the subject reach a peak and are declining in response to thetemozolomide or the pharmaceutically acceptable salt.
 16. The method ofclaim 10 wherein the EGFRvIII peptide is administered after T_(REG)cells in the subject reach a peak and are declining in response to thetemozolomide or the pharmaceutically acceptable salt.
 17. The method ofclaim 1 wherein the peptide and the temozolomide or salt thereof areadministered repeatedly in a cycle.
 18. The method of claim 10 whereinthe peptide and the temozolomide or salt thereof are administeredrepeatedly in a cycle.
 19. The method of claim 1 wherein the combinedadministration induces a DTH response in the subject.
 20. The method ofclaim 10 wherein the combined administration induces a DTH response inthe subject.
 21. The method of claim 1 wherein EGFRvIII peptide-specificγ-IFN producing CD8⁺ T cells are induced in the subject.
 22. The methodof claim 10 wherein EGFRvIII peptide-specific γ-IFN producing CD8⁺ Tcells are induced in the subject.
 23. The method of claim 1 wherein anIgG response to EGFRvIII peptide is induced in the subject.
 24. Themethod of claim 1 wherein an IgG response to EGFRvIII peptide is inducedin the subject.